Because opioids can act directly on certain peripheral tissues, to produce antinociception and analgesia, peripheral administration of opioids offers a promising strategy to avoid central nervous system side-effects that limit the use of systemic opioids. However, the effectiveness of peripherally administered opioids may be limited by the development of tolerance. We propose to investigate the basic mechanisms of tolerance and physical dependence of mu-opioids to produce local peripheral antinociception (i.e., inhibition of inflammatory mediator-induced hyperalgesia). We will determine the dose dependence and time-course of tolerance to mu-opioids injected intradermally in the dorsum of the hindpaw using behavioral methods. In addition, we will investigate dependence by testing for withdrawal hyperexcitability induced by either abstinence or naloxone. Since increased excitability of sensory nerve fibers is an important mechanism underlying hyperalgesia and since opioids act in the periphery to inhibit sensitization of primary afferent nociceptors, we will also use electrophysiological methods to investigate whether the antinociceptive effects of opioids on primary afferent nerve fibers are subject to tolerance and dependence. Through the use of cross- tolerance experiments (with non-opioid peripherally-acting antinociceptive agents, A/1-adenosine and alpha/2-adrenergic agonists) we plan to demonstrate that both the development of tolerance and the development of dependence to mu-opioid receptor depends on changes in intrathecal second messenger pathways downstream to the mu-opioid receptor. Finally, we will use cross-tolerance experiments in combination with the molecular biological ablation of mu-opioid receptors on the primary afferent to determine if the mu-opioid receptor involved in the development of tolerance is on the primary afferent neuron. These experiments should provide an understanding of the basic mechanisms underlying tolerance to the antinociceptive action of opioids in the periphery. This information may provide a rational basis for the development of strategies to maximize the clinical efficacy of local peripherally administered mu-opioids or other peripherally-acting analgesics.